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Polarity of IAA Effect on Sieve-Tube and Xylem Regeneration in Coleus and Tomato Stems.

A technique is described for the processing of regenerated xylem and sieve tubes from the same wound area for microscopic and quantitative comparison.Regeneration was examined in internodes of 2 developmental stages in Coleus: internode 2, elongating, characteristic of primary growth; and internode 5, non-elongating, characteristic of secondary growth.Transport of indoleacetic acid (IAA) in excised number 5 internodes of Coleus is strictly polar, in a basipetal direction, judging by a regeneration bioassay involving both sieve tube strands and xylem cells. Similar results were obtained with tomato.If isolated number 5 Coleus internodes are not treated with hormone, they regenerate no xylem cells and a small number of sieve tube strands. With increasing concentrations of IAA added apically, the number of regenerated sieve tube strands (and, with higher concentrations, of xylem cells) increases progressively up to 1% IAA, the highest concentration applied.Internode 2 of Coleus regenerates fewer xylem cells or sieve tube strands than internode 5, whether on the otherwise intact plant or with a given concentration of IAA added apically. The amount of regenerated xylem increases with added apical IAA, except that the highest concentration gives no further increase. The number of xylem cells regenerated in intact plants occurs at the same interpolated IAA concentration as in number 5 internodes. No concentration of IAA tried provided replacement of intact number of sieve tube strands in internode 2.IAA can exert a regenerative stimulus on both xylem and sieve tubes in the area immediately adjacent to the site of its application.

Journal Article↗

The role of cytokinin in sieve tube regeneration and callose production in wounded coleus internodes.

Cytokinin proved to be a controlling factor in sieve tube regeneration around wounded collateral bundles in an in vivo system in which the endogenous cytokinin level had been minimized. Both kinetin and zeatin were applied in aqueous solution to the bases of excised, mature internodes of Coleus blumei Benth. that had an active vascular cambium. Each internode also received indoleacetic acid (IAA) in lanolin at its apical end. Under either low (0.1% w/w) or high (1.0% w/w) auxin concentrations, the control internodes (without exogenous cytokinin) exhibited small amounts of sieve tube regeneration. At appropriate concentrations, both kinetin and zeatin induced a significant increase in sieve tube regeneration around the wound. However, the highest concentration of kinetin tested (50 mug/mL) completely inhibited this process. Kinetin was the most effective with high auxin (1.0% IAA), while zeatin was the most effective with low auxin level (0.1% IAA). Kinetin and zeatin showed the strongest promotive effect at 10 mug/mL and 20 mug/mL, respectively. Both cytokinins also induced supplementary phloem regeneration further from the wound surface. In addition to their effects on vascular tissue regeneration, both cytokinins promoted callose production. This was most evident on the sieve plates of the regenerated sieve tube members and on the walls of the parenchyma cells around the wound. The largest deposits of callose were found in both regenerated sieve tube members and parenchyma cells at the highest cytokinin concentration tested (50 mug/mL). The possible role of cytokinin in controlling callose accumulation in the sieve tubes during autumn is discussed.

Journal Article↗

Thioredoxin regenerates proteins inactivated by oxidative stress in endothelial cells.

The thioredoxin/thioredoxin reductase system has been studied as regenerative machinery for proteins inactivated by oxidative stress in vitro and in cultured endothelial cells. Mammalian glyceraldehyde-3-phosphate dehydrogenase was used as the main model enzyme for monitoring the oxidative damage and the regeneration. Thioredoxin and its reductase purified from bovine liver were used as the regenerating system. The physiological concentrations (2-14 microM) of reduced thioredoxin, with 0.125 microM thioredoxin reductase and 0.25 mM NADPH, regenerated H2O2-inactivated glyceraldehyde-3-phosphate dehydrogenase and other mammalian enzymes almost completely within 20 min at 37 degrees C. Although the treatment of endothelial cells with 0.2-12 mM H2O2 for 5 min resulted in a marked decrease in the activity of glyceraldehyde-3-phosphate dehydrogenase, it had no effect on the activities of thioredoxin and thioredoxin reductase. Essentially all of the thioredoxin in endothelial cells at control state was in the reduced form and 70-85% remained in the reduced form even after the H2O2 treatment. The inactivated glyceraldehyde-3-phosphate dehydrogenase in a cell lysate prepared from the H2O2-treated endothelial cells was regenerated by incubating the lysate with 3 mM NADPH at 37 degrees C and the antiserum raised against bovine liver thioredoxin inhibited the regeneration. The inhibition of thioredoxin reductase activity by 13-cis-retinoic acid resulted in a decrease in the regeneration of glyceraldehyde-3-phosphate dehydrogenase in the H2O2-treated endothelial cells. The present findings provide evidence that thioredoxin is involved in the regeneration of proteins inactivated by oxidative stress in endothelial cells.

Animals↗

In vitro ATP regeneration from polyphosphate and AMP by polyphosphate:AMP phosphotransferase and adenylate kinase from Acinetobacter johnsonii 210A.

In vitro enzyme-based ATP regeneration systems are important for improving yields of ATP-dependent enzymatic reactions for preparative organic synthesis and biocatalysis. Several enzymatic ATP regeneration systems have been described but have some disadvantages. We report here on the use of polyphosphate:AMP phosphotransferase (PPT) from Acinetobacter johnsonii strain 210A in an ATP regeneration system based on the use of polyphosphate (polyP) and AMP as substrates. We have examined the substrate specificity of PPT and demonstrated ATP regeneration from AMP and polyP using firefly luciferase and hexokinase as model ATP-requiring enzymes. PPT catalyzes the reaction polyP(n) + AMP --> ADP + polyP(n-1). The ADP can be converted to ATP by adenylate kinase (AdK). Substrate specificity with nucleoside and 2'-deoxynucleoside monophosphates was examined using partially purified PPT by measuring the formation of nucleoside diphosphates with high-pressure liquid chromatography. AMP and 2'-dAMP were efficiently phosphorylated to ADP and 2'-dADP, respectively. GMP, UMP, CMP, and IMP were not converted to the corresponding diphosphates at significant rates. Sufficient AdK and PPT activity in A. johnsonii 210A cell extract allowed demonstration of polyP-dependent ATP regeneration using a firefly luciferase-based ATP assay. Bioluminescence from the luciferase reaction, which normally decays very rapidly, was sustained in the presence of A. johnsonii 210A cell extract, MgCl(2), polyP(n=35), and AMP. Similar reaction mixtures containing strain 210A cell extract or partially purified PPT, polyP, AMP, glucose, and hexokinase formed glucose 6-phosphate. The results indicate that PPT from A. johnsonii is specific for AMP and 2'-dAMP and catalyzes a key reaction in the cell-free regeneration of ATP from AMP and polyP. The PPT/AdK system provides an alternative to existing enzymatic ATP regeneration systems in which phosphoenolpyruvate and acetylphosphate serve as phosphoryl donors and has the advantage that AMP and polyP are stabile, inexpensive substrates.

Acinetobacter↗

Programmed macromolecular synthesis in regenerating karyoplasts.

Conditions for the preparation, purification, and maintenance of karyoplasts which could regenerate to reform whole viable cells were defined. Results of biochemical analyses of such karyoplasts at various times during regeneration indicated that a reproducible biosynthetic program was followed. Thus, an examination of the polypeptides made during regeneration by two-dimensional gel electrophoresis showed that the pattern of radiolabeled polypeptides synthesized at each time studied was specific and was significantly different from that observed at other times during regeneration. Polypeptides associated with three major cellular fractions--nuclear, cytoskeletal-microtrabecular, and soluble--were among the most dramatically regulated molecules. Other polypeptides, such as the major components of microfilaments and intermediate filaments, were synthesized at relatively constant rates and were assembled into structures throughout regeneration. Likewise, microtubules appeared to be reformed throughout regeneration, even in the absence of identifiable centriole-associated organizing centers. Finally, analysis of DNA synthesis by autoradiography showed that, even when prepared from whole cells synchronized at the G1/S interface, karyoplasts could not begin making DNA until they had regenerated an almost complete complement of cytoplasm.

Animals↗

The in vivo regeneration of goldfish rhodopsin and porphyropsin.

Goldfish with retinas rich in either rhodopsin or porphyropsin were illuminated with bright light and then placed in the dark room to allow visual pigment regeneration. The kinetics of this in vivo pigment regeneration were followed by sampling these animals at regular time intervals. The first-order kinetic rate constant for the initial period of porphyropsin regeneration at 20 degrees C was 8.3 X 10(-3) nmol kg-1 body weight min-1 and the half-life of this reaction was 83 min. At 30 degrees C, the rate constant was increased to 1.4 X 10(-2) nmol kg-1 body weight min-1, yielding a reduced half-life of 49 min. This suggests that the Q10 of porphyropsin regeneration is about 1.7. In goldfish retinas enriched with rhodopsin (62% rhodopsin and 38% porphyropsin), the initial phase of visual pigment regeneration (at 30 degrees C) proceeded at a slower rate (first-order rate constant: 6.5 X 10(-3) nmol kg-1 body weight min-1; half-life of reaction = 106 min) than the rate of porphyropsin regeneration. This suggests that the high proportion of rhodopsin in the retina of goldfish held at 30 degrees C is not a direct result of a faster rate of regeneration of rhodopsin than of porphyropsin.

Adaptation, Ocular↗

Heterogeneity in regeneration of bacteriorhodopsin from bacterio-opsin and all-trans retinal at high temperatures: implications for dynamic structural fluctuations.

Measurements of regeneration kinetics were performed in order to investigate the regeneration mechanisms of bacteriorhodopsin (bR) from thermally unfolded bacterio-opsin (bO) and all-trans retinal. Regeneration kinetics data were successfully fitted to a single exponential function when regeneration was performed at 25 degrees C after incubation at high temperatures. Conversely, the process of regeneration after the addition of retinal to bO at high temperatures occurred at two different rate constants. These findings strongly suggest that the slower regeneration of bR at high temperatures occurs as a result of dynamic structural fluctuation of bO, whereas the faster process corresponds to regeneration from bO, which retains a native structure capable of retinal binding.

Bacteriorhodopsins↗

Acuity recovery and cone pigment regeneration after a bleach in patients with retinitis pigmentosa and rhodopsin mutations.

PURPOSE: To assess visual acuity recovery times and cone photopigment regeneration kinetics after a bleach in the fovea of patients with dominant retinitis pigmentosa due to rhodopsin mutations. METHODS: The authors measured acuity recovery times by computerized photostress testing in 13 patients with dominant retinitis pigmentosa and one of eight rhodopsin mutations. The authors also measured their time constants of cone photopigment regeneration with a video imaging fundus reflectometer to determine whether acuity recovery time depended on pigment regeneration kinetics. These values were compared with those of normal subjects, by the Mann-Whitney U test. The relationship between acuity recovery time and the time constant of cone photopigment regeneration among the patients was quantified by the Spearman rank correlation. RESULTS: The visual acuity recovery times, which averaged 22.0 seconds for the patients with retinitis pigmentosa and 11.2 seconds for the normal subjects, were significantly slower for the patient group (P < 0.001). The time constants of cone pigment regeneration, which averaged 172 seconds for the patients with retinitis pigmentosa and 118 seconds for the normal subjects, also were significantly slower for the patient group (P = 0.043). The authors also found a significant, positive correlation between the visual acuity recovery time and the time constant of pigment regeneration for the patients with retinitis pigmentosa (r = 0.65, P = 0.017). CONCLUSIONS: A slowing of foveal visual acuity recovery and cone pigment regeneration, which are related to each other, can occur in patients with retinitis pigmentosa, due to a rod-specific gene defect.

Adult↗

Axonal regeneration of mouse retinal ganglion cells by peripheral nerve transplantation; a quantitative study.

Purpose: Retinal ganglion cells (RGCs) of adult mammals can regenerate their axons along a segment of the peripheral nerve (PN) that is transplanted to the cut optic nerve. There have been many trials of PN transplantation to induce axonal regeneration of RGCs in adult rodents, cats and ferrets. However, because of the technical difficulty in transplant operation, PN transplantation in adult mice has not been carried out in spite of the availability of many kinds of gene-manipulated animals. Here we report the procedures for successful PN transplantation in this species. Methods: We made intraretinal (IR) and retrobulbar (RB) approaches for PN transplantation. Four weeks after PN transplantation, RGCs with regenerated axons were identified by retrograde labeling with rhodamine or horseradish peroxidase applied into the PN segment. Results: A quantitative survey showed that the mean regeneration ratio was 1.0 % (n = 8) in IR transplantation, whereas it was only 0.1 % in RB transplantation (n = 11). As previously shown in other species, the regenerated RGCs were predominantly larger-bodied cells in com-parison to intact cells. Conclusion: Possible reasons for the difference in regeneration ratio between the two transplant approaches and the feature of soma size of regenerated RGCs are discussed.

Journal Article↗

Stimulation of Schwann cell proliferation and axonal regeneration by FK 506.

PURPOSE: Nerve allografts are highly antigenic and, thus, require the continuous use of immunosuppressive drugs. FK 506 was found to pre-vent rejection successfully. However, clinically neurotoxic complications have been noted in the central and peripheral nervous system although an increased rate of axonal regeneration has also been shown after nerve crush experiments. To investigate whether a possible regeneration pro-moting potency of FK 506 is determined via an influence on Schwann cells, Schwann cells were cultured from the sciatic nerve of the rat. METHODS: The effect of 100 micro M FK 506 administered daily on these cultures was assessed microscopically over a period of seven days and compared to an untreated control group of cultures. Additionally, the changes in intracellular calcium were recorded using a laser scanning microscope. In vivo regeneration of autologous rat sciatic nerve grafts was assessed clinically, histologically and morphometrically after two and six weeks. The animals received a daily administration of 0.6 mg FK 506/kg body weight, the control received saline. RESULTS: In vitro FK 506 increased the Schwann cell number in culture significantly compared to non treated cultures, while the fibrocyte population was decreased. FK 506 caused a transient increase of intracellular calcium levels in cultured cells. In vivo, a significantly higher axon count was observed in the FK 506 treated grafts after two weeks regeneration compared with controls. Good regeneration was noted in all grafts after six weeks regeneration. CONCLUSIONS: The increased axon counts and decreased myelin debris in the FK 506 grafts after two weeks indicate an accelerated Wallerian degeneration and increased axon sprouting into the graft initially. The inhibition of calcineurin activity is not the mediator of the neurotrophic effect. FK 506 promotes axonal regeneration through binding to FKBP-12. The increase of intracellular calcium may induce Schwann cell pro-liferation via calmodulin. The therapeutic relevance for autologous nerve grafting, however, has to be defined in further studies.

Journal Article↗

[The effect of the duration of callus culture on the accumulation of genetic alterations in regenerated pea Pisum sativum L].

Two groups of regenerant plants were obtained from different pea genotypes (lines R-9 and W1 and cultivar Viola). The first group was derived after eight months of culture and the second, from calluses cultured for a prolonged (more than ten years) time. Using random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) methods, the regenerants and the original lines were compared with regard to genetic differences. The regenerants from both groups were shown to differ in DNA polymorphism from the original lines and from one another. The divergence of the regenerants was also different, depending largely on the original genotype. Examination of genetic differences between the first and the second group showed that the variability increased with culturing time. This was particularly evident for regenerants of the Viola cultivar, in which variability ranged from 0-5% (first group of regenerants) to 10% (second group of regenerants).

DNA, Plant↗

Ascorbic acid regeneration in chromaffin granules. In situ kinetics.

We have investigated in intact chromaffin secretory vesicles the kinetics, specificity, and mechanism of intragranular ascorbic acid regeneration by extragranular ascorbic acid. The apparent Km of internal ascorbic acid regeneration for external ascorbic acid was 280 microM by Lineweaver-Burk analysis and 287 microM by Eadie-Hofstee analysis. Intragranular ascorbic acid regeneration was specifically mediated by extragranular ascorbic acid or its isomer isoascorbic acid; the reducing agents glutathione, thiourea, homocysteine, NADH, and NADPH did not support regeneration. The structural analog D-glucose did not inhibit regeneration by external ascorbic acid, suggesting specificity at the membrane site of electron transfer. The driving force for regeneration of intragranular ascorbic acid was independent of membrane potential, absolute intragranular and extragranular pH, and ATPase activity, but might be coupled to the pH difference across the chromaffin granule membrane. Since the apparent Km of regeneration was approximately 10-fold below the cytosolic concentration of ascorbic acid, the reaction may proceed at Vmax in situ.

Adenosine Triphosphatases↗

[Regeneration of Nocardia orientalis protoplasts].

A method for effective regeneration of the protoplasts of N. orientalis, a vancomycin-producing organism into viable cells on a rich organic medium was developed. The dependence of the regeneration on the conditions of the protoplast plating out and the level of the regeneration medium dehydration was studied. The highest positive effect was observed when the protoplasts were suspended in the agarized medium and then plated out on the regeneration medium dehydrated by 2.5 per cent. The frequency of the protoplast regeneration increased on addition of bovine serum albumin to the regeneration medium. The effect of bovine serum albumin depended on its concentration. When the albumin concentration was optimal (0.01 per cent) the regeneration amounted to 100 per cent.

Culture Media↗

Hypoxia inhibits rhodopsin regeneration in the excised mouse eye.

PURPOSE: The goal of this study was to evaluate the effect of oxygen deprivation on rhodopsin regeneration in the excised mouse eye. METHODS: A new preparation for studying rhodopsin regeneration with a superfused excised albino mouse eye was developed. The preparation exhibits multiple regenerations after moderate bleaches (15%-20%) and is sensitive to the composition of the perfusate, allowing reversible testing of conditions. A variety of protocols were used to evaluate the effect of hypoxia. The major experiments varied the timing of the decreased oxygen relative to the illumination: Decreasing the oxygen (1) before the illumination, (2) immediately after the illumination, or (3) some time after the illumination, after half of the rhodopsin had regenerated. Elevated concentrations of extracellular glucose also were used for certain experiments. RESULTS: Regardless of protocol, at low levels of oxygen no rhodopsin regeneration was observed. The effects were reversible, however, and decreased oxygen for up to 3 hr did not diminish the ability of the eye to regenerate rhodopsin after restoration of the oxygen and a subsequent bleach. CONCLUSIONS: Low levels of oxygen eliminated rhodopsin regeneration in the excised eye. The effect could not be offset by high levels of glucose, illumination, or other conditions known to reduce the dependence of photoreceptors on aerobic respiration.

Animals↗

Gangliosides stimulate protein synthesis, growth, and axon number of regenerating limb buds.

When a newt limb is amputated and begins to regrow, regenerating axons exert a neurotrophic influence on the limb regeneration process. Previous studies have shown that direct manipulation of limb nerves, by electrical stimulation or a conditioning lesion, elevates protein synthesis, increases neurotization and accelerates growth of the limb bud. Since exogenously supplied gangliosides accelerate axonal sprouting in regenerating nerves, we wanted to know whether gangliosides would similarly affect limb regeneration. To test this, regrowing limb buds were either infused with or immersed into gangliosides, or animals were injected intraperitoneally with gangliosides. Infused gangliosides elevated protein synthesis in limb buds 15% and increased the number of axons in limb buds 45% by 6 hours after infusion. Regenerating limb bud morphogenesis was initiated 3-4 days earlier in animals receiving i.p. injections of gangliosides every 12 hours. Similarly, limbs immersed daily in gangliosides began regrowth sooner than contralateral controls and this advantage was maintained throughout the period of observation. These findings indicate that treatment with gangliosides has a salutary effect on limb regeneration.

Animals↗

The interaction of nerves of opposite regenerating polarity in fused newt forelimbs.

Previous studies involving nerve interactions and limb regenerative processes were carried out on adult newts after their forelimbs were amputated through the distal radius and ulna and fused end-to-end. On the basis of limb regeneration results at the junction of the fused limbs, it was postulated that regenerating nerves from each limb (i.e., nerves of opposite polarity) would not invade the foreign territory of the contralateral limb if it were already normally innervated. A direct study of this nerve interaction, however, was not made in this earlier study. The present investigation was designed to obtain direct histological and electrophysiological evidence for the interaction of nerves of opposite regenerating polarity in fused newt forelimbs. The primary objective was to determine how the regenerating nerves would interact in the establishment of innervation territories-first, at the fusion zone, which represents the junction of the normal innervation territories of the nerves of each limb; and secondly, half way up one of the limbs, where interaction would occur in a territory normally innervated by only one of the regenerating nerves. The results showed that when nerves of opposite regenerating polarity approached one another at the junction of the fused limbs a discontinuation of axonal growth occurred; no indication of overlap of nerves into foreign territory was seen. When the nerves were allowed to interact within one of the fused limbs, however, an overlap of nerve fibers and a functional "double innervation" of that limb was demonstrated. These results are discussed in terms of possible mechanisms for the establishment of innervation territories in salamander limbs. The question of nerve-muscle reinnervation specificity is also raised.

Animals↗

Stem cells in CNS and cardiac regeneration.

The central nervous system (CNS) and the heart muscle regenerate poorly after injury, yet evidence is mounting that both harbor cells capable of rebuilding neural and cardiac tissue. The reason for the poor regenerative response of CNS tissue and myocardium must therefore lie in the nature of the injury environment, which promotes fibrosis over regeneration. Strategies for regenerating these tissues thus rely on overcoming the fibrotic response by filling lesions with tissue-specific regeneration-competent cells that replace or rescue dying cells, or by activating endogenous regeneration-competent cells that do likewise. There has also been considerable excitement about the possibility of transplanting bone marrow cells into CNS or cardiac lesions to repair them, because bone marrow cells have been reported to be pluripotent. In this chapter, contemporary evidence for the existence of regeneration-competent cells in the CNS and heart is discussed, as well as attempts to use these cells and bone marrow cells to reconstitute new tissue.

Cardiac Surgical Procedures↗

Immunocytochemical study of extracellular matrix components during lens and neural retina regeneration in the adult newt.

We have conducted an immunocytochemical study of fibronectin, laminin, heparan sulfate proteoglycans and nidogen-entactin during lens and neural retina regeneration in the adult newt from 0 to 60 days. In the normal eye, fibronectin was detected in the corneal stroma and Descemet's membrane, in dorsal and ventral irises and lens capsule but not in Bowman's membrane of the cornea. In normal neural retina, fibronectin was found in Bruch's and inner limiting membranes. Heparan sulfate proteoglycans gave a slight signal in both irises and the lens capsule. Nidogen-entactin distribution in the cornea was similar to that of fibronectin; it was absent from the stroma of both irises, and the signal was weak in the pigmented iris epithelium. Nidogen-entactin was not detected in the lens capsule and inner limiting membrane of the neural retina but was present in Bruch's membrane. During the first 15 days of lens regeneration, fibronectin and nidogen-entactin decreased but did not disappear from the pupillary margin of both irises, and no signal was obtained for laminin and heparan sulfate proteoglycans. From day 15 to day 60 fibronectin and nidogen-entactin increased in both irises and lens capsule. The signal for laminin was restricted to the lens capsule. Heparan sulfate proteoglycans gave a slight signal in both irises and in the lens capsule. During the first 25 days of neural retina regeneration, fibronectin was the first to appear in Bruch's membrane and the cell border of the new neuroepithelium and remained during the entire process. Laminin appeared after 41 days in the inner limiting and Bruch's membranes, but by day 50 it appeared as a weak signal only in the inner limiting membrane. Heparan sulfate proteoglycans were not detected at any of the regeneration stages studied. Nidogen-entactin was only detected in Bruch's membrane and around the cells and blood vessels of the new neural retina. Later it was detected in the inner limiting membrane but not in Bruch's membrane. Thus, the results obtained showed that extracellular matrix components do change during both lens and neural retina regeneration. These changes may play an important role during both regenerating processes.

Animals↗